The introduction of an aminoalkylamide into the C-terminal end of a biologically active peptide has in some cases had beneficial effects on the metabolic stability and activity (EP-A 179 332). The preparation of the peptides modified in this way has made use of the classical coupling of fragments in solution.
EP-A 179 332 reports the observation that simple dipeptides such as:
benzyloxycarbonyl-Lys-Phe-OMe PA1 benzyloxycarbonyl-D-Lys-Phe-NH-(CH.sub.2).sub.8 -NH.sub.2.2HCl PA1 EP-A 179 332 discloses novel peptides having an aminoalkylamino group bound to the C-terminal end of the peptide. These peptides are active on the central nervous system and act on the cholinergic system. As described in EP-A 179 332, these peptides effect a significant, dose-dependent weakening of the amnesia induced by electric shock or scopolamine in mice ("one-trial passive avoidance test"). The minimum effective dose, for example, required for the compound PA1 Met(O)-Glu-His-Phe-D-Lys-Phe-NH-(CH.sub.2).sub.8 -NH.sub.2 PA1 In humans, the peptides of EP-A 179 332 have a mood-lightening, antidepressant and anxiolytic action. These peptides increase attention to the environment, improve learning and memory performance, have a favorable effect on resocialization processes and can be used for all diseases of post-traumatic and degenerative brain damage which are associated with a reduced central acetylcholine metabolism function, for example, mild dementia and also early manifestations of Alzheimer's disease and the like. PA1 B represents one or more, identical or different, amino acids, PA1 X denotes (C.sub.1 -C.sub.12)-alkylene or (C.sub.6 -C.sub.10)-aryl-(C.sub.1 -C.sub.12)alkylene, PA1 Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 are identical or different and denote hydrogen methyl, methoxy or nitro, at least one of these radicals denoting hydrogen, PA1 V denotes hydrogen or a carboxyl protective group, PA1 W denotes --[CH.sub.2 ].sub.n -- or --O--[CH.sub.2 ].sub.n --, PA1 m is 0 or 1, PA1 n is an integer from 0 to 6, and PA1 p is an integer from 0 to 5. PA1 a) reaction of a compound of-the formula II ##STR4## in which R represents a leaving group which can be detached nucleophilically, PA1 V represents a carboxyl protective group, and PA1 W, Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 and are as defined above, with a compound of the formula III EQU A--[B].sub.p --NH--[X].sub.m --NH.sub.2 (III) PA1 in which A represents an amino protective group which is labile to bases or labile to weak acids, and B, X, p and m are as defined above, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I, with the formation of the free NH.sub.2 and/or CO.sub.2 H group(s), the preferred processes being those in which V is selectively eliminated, for example by reductive cleavage with Zn/glacial acetic acid, or PA1 b) reaction of a compound of the formula I in which A denotes hydrogen, and B, X, Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, V, W, m, n and p are as defined above, with a compound of the formula IV EQU A--[B].sub.5-p --OH (IV) PA1 in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group.
cause the urge to groom and move in rats following intracerebroventricular (i.c.v.) administration of 10 .mu.g. At the same time, cholinergic mechanisms in the central nervous system (CNS) are influenced. In the striatum of rats, the choline content is increased following subcutaneous (s.c.) administration of 10 .mu.g. The effects are intensified if the carboxyl groups carries a radical with basic substituents, it being possible for lysine to be in the D-form. Thus, for example, the compound
shows the same action on i.c.v. or s.c. administration of 1 .mu.g. The effects described are characteristic of ACTH and MSH.
is 0.03 .mu.g/kg, following s.c. administration.
In the solid-phase synthesis of peptides (see Patchornik, Cohen in Perspectives in Peptide Chemistry, pages 118-128 (Karger, Basle 1981)) the reactive chains are often not grafted directly onto the synthetic resin material, but are bonded to the carrier material by what are called spacers or links. The literature (for example Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101-117 (Karger, Basle 1981)) discloses, for example, reagents for introducing such spacers (called "linkage agents") which have the formulae VI, VII and VIII. ##STR2##
New linkage agents which allow direct construction, by solid phase synthesis, of peptides modified by C-terminal aminoalkylamide or hydrazide have been found.
Thus, the present invention relates to compounds of the formula I ##STR3## in which A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids,
The present invention relates to new linkage agents or anchor groups of the formula I. These compounds of formula I allow direct construction, by solid phase synthesis, of peptides modified by C-terminal amino-alkylamide or hydrazide. In the solid phase synthesis of peptides, the reactive amino acid chains are often not grafted directly onto the synthetic resin material, but are bonded to the carrier material by what are called linkage agents, anchor groups, or spacers. The compounds of formula I can be used in the synthesis of known or new peptides wherein they function as an anchor group during the solid phase synthesis of a peptide, then upon cleavage release a peptide having an aminoalkylamide or hydrazide end group. The Examples, infra, demonstrate the solid phase synthesis of such new useful peptides employing an anchor or spacer group according to formula I.
Preferred compounds of the formula I are those in which p is 0, 1 or 2, in particular, 0, and/or in which m is 1. Thus, for example, when p is 0, no amino acid B is present and when p is 1 only one amino acid B is present.
X is preferably --[CH.sub.2 ].sub.q --, it being possible for q to be 1-12, preferably 1-8.
Preferably at least 2, in particular at least 3, of the radicals Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 denote hydrogen.
Protective groups which are labile to bases or labile to weak acids are, in particular, urethane protective groups, such as Fmoc, Ddz, Bpoc, Msc, Peoc, Pse and Tse, preferably Fmoc (see, for example, Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23).
B represents one or more amino acids, preferably an .alpha.-amino acid, which, if chiral, can be present in the D or L form. When more than one amino acid is present, the amino acids are bound together by amide linkages as is known in amino acid and peptide chemistry. Preference is given to naturally occurring amino acids, their enantiomers, homologs, derivatives and simple metabolites, e.g. hydroxyproline, citruline, 4-hydroxymethylproline, N-.epsilon.-hydroxylysine or 4-methylglutamine acid. (See further, for example, Wunsch et al., Houben-Weyl 15/1 and 2, Stuttgart, Thieme 1974). The abbreviations used for the amino acids correspond to the three-letter code which is customary in peptide chemistry, and described in, for example, Europ. J. Biochem. 138, 9 (1984). Thus, for example, the following amino acids are suitable:
Aad, Abu, .gamma. Abu, ABz, 2ABz, .epsilon. Aca, Ach, Acp, Adpd, Ahb, Aib, .beta. Aib, Ala, .beta. Ala, .DELTA. Ala, Alg, All, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gln, Glu, Gly, Guv, hCys, His, hSer, Hyl, Hyp, 3Hyp, Ile, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, .beta. Lys, .DELTA. Lys, Met, Mim, Min, nArg, Nle, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, .DELTA. Pro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, .beta. Thi, Thr, Thy, Thx, Tia, Tle, Tly, Trp, Trta, Tyr, Val and the residues of the corresponding enantiomeric D-amino acids.
Functional groups in the side chains of the said amino acids can be in protected form. Suitable protective groups are described in Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23, and in Bullesbach, Kontakte (Merck) 1980, No. 1, pages 23-35. The preferred groups are those which are stable to bases and weak acids and can be eliminated using strong acids.
Alkylene can be straight-chain or branched. Examples of definitions of (C.sub.6 -C.sub.10)-aryl are phenyl, tolyl or naphthyl; phenyl is preferred.
A carboxyl protective group V is, for example, (C.sub.1 -C.sub.6)-alkyl or (C.sub.7 -C.sub.11)-aralkyl; preference is given to methyl, ethyl, tert.butyl, benzyl and modified benzyl, such as p-chloro-, p-bromo-, p-nitro- and p-methoxybenzyl and the nitrogen analog picolyl. In the wider sense, such protective groups include activated ester groups such as ONSu, OBt, OObt or p-nitrophenoxy.
The invention also relates to a process for the preparation of the compounds of the formula I, which comprises
A leaving group R which can be detached nucleophilically is, for example, halogen, such as chlorine, bromine and iodine, or activated aryloxy, such as p-nitrophenoxy.
The reaction of a compound of the formula II with a compound of the formula III is preferably carried out in an aprotic solvent such as, for example, THF, DMF, CHCl.sub.3 or CH.sub.2 Cl.sub.2, advantageously in the presence of a base such as, for example, a tertiary amine, for example ethyl triisopropylamine, triethylamine or pyridine, the addition of an acylation catalyst such as, for example, DMAP, HOObt or HOBt having an advantageous effect, at a temperature between 0.degree. C. and the boiling point of the reaction mixture, preferably between 0.degree. C. and 40.degree. C.
Compounds of the formula I (A=hydrogen) are reacted with compounds of the formula IV, their active ester, halide or azide preferably in an organic solvent, such as DMF, advantageously in the presence of a base such as, for example, a tert.amine, at a temperature between -10.degree. C. and the boiling point of the reaction mixture, preferably at room temperature. Examples of suitable active esters are the ONSu, OBt, OObt and p-nitrophenoxy compounds. Preferred halogen derivatives are the chlorides. Pyridinium perchlorate can be added to improve the solubility.
Compounds of the formula II are prepared by, for example, reacting esters of the formula IX ##STR5## in which Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, W and V are as defined above, but V does not denote hydrogen, with phosgene or phosgene derivatives such as, for example, nitrophenyl chloroformate in an aprotic polar solvent, for example THF or DMF, mixed with a tert. base, for example a tert. amine such as pyridine, preferably in the ratio 1:1, at a temperature between -40.degree. C. and room temperature, preferably between -20.degree. C. and 0.degree. C.
The invention also relates to the use of a compound of the formula I, in which V denotes hydrogen and A does not denote hydrogen, in the solid-phase synthesis of compounds of the formula V: EQU P--NH--[X].sub.m --NH.sub.2 (V)
in which P represents a peptide residue comprising q.ltoreq.p+1 .alpha.-amino acids, and X, m and p are as defined above, and to a process for the preparation of a peptide of the formula V, in which P, X, m and p are as defined above, by solid-phase synthesis, which comprises coupling a compound of the formula I, in which A does not denote hydrogen, and V represents hydrogen, to a resin, eliminating the protective group A, stepwise coupling on q-p .alpha.-amino acids which are, where appropriate, in the form of their activated derivatives and which have been temporarily protected by amino protective groups which are labile to bases or labile to weak acids and, after construction is complete, liberating the peptide from the resin by treatment with a moderately strong to strong acid, the temporarily introduced side-chain protective groups being eliminated again at the same time or, by suitable measures, subsequent thereto.
If necessary to prevent side reactions or for the synthesis of specific peptides, the functional groups in the side chain of amino acids are additionally protected by suitable protective groups (see, for example, T. W. Greene, "Protective Groups in Organic Syntheses", New York, John Wiley & Sons, 1981), those primarily used being Arg(Tos), Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBut), Cys(4-MeBzl), Cys(Acm), Cys(SBut), Glu(OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-2), Lys(Boc), Met(O), Ser(Bzl), Ser(But), Thr(Bzl), Thr(But).
The resins used as carrier material are commercially available. BHA and MBHA resins are preferred.
The peptide of the formula V is then cleaved off by treatment with the moderately strong to strong acids customarily used in peptide synthesis (for example trifluoracetic acid and HF), there being cleavage of the urethane protective group contained in the spacer.
It is possible to use as coupling reagent for the compound of the formula I (V=H) and the other amino acid derivatives all possible activating reagents used in peptide synthesis, see, for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry), volume 15/2, but in particular carbodiimides such as, for example, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. This coupling can be carried out directly by addition of the amino acid derivative with the activating reagent and, where appropriate, an additive which suppresses racemization, such as, for example, 1-hydroxybenzotriazole (HOBt) (W. Konig, R. Geiger, Chem. Ber. 102., 708 (1970)) or 3-hydroxy-4-oxo-3,4-dihydroxybenzotriazine (HOOBt) (W. Konig, R. Geiger, Chem. Ber. 103, 2054 (1970)) to the resin, or the preactivation of the amino acid derivative can be carried out separately as the symmetric anhydride or HOBt or HOObt ester, and the solution of the activated species in a suitable solvent can be added to the peptide-resin which is ready for coupling.
The coupling and activation of the compound of the formula I (V=H) and of the amino acid derivatives with one of the abovementioned activating reagents can be carried out in dimethylformamide or methylene chloride or a mixture of the two. The activated amino acid derivative is normally used in a 1.5- to 4-fold excess. In cases where incomplete coupling occurs, the coupling reaction is repeated, without previously carrying out the deblocking of the .alpha.-amino group of the peptide-resin which is necessary for coupling the next amino acid in the sequence.
Successful completion of the coupling reaction can be checked using the ninhydrin reaction as described, for example, by E. Kaiser et al. Anal. Biochem. 34,595 (1970).
The synthesis can also be carried out automatically, for example using an Applied Biosystems model 430A peptide synthesizer, it being possible to use either the synthesis programs provided by the apparatus manufacturer or those constructed by the user himself. The latter are particularly employed when amino acid derivatives protected with the Fmoc group are used.
When the peptide amides are cleaved off the resin with hydrogen fluoride and trifluoroacetic acid, it is customary to add substances as cation traps, such as phenol, cresol, thiocresol, thioanisole, anisole, ethanedithiol, dimethyl sulfide, ethyl methyl sulfide or a mixture of two or more of these auxiliaries. In this connection, the trifluoroacetic acid can also be used diluted by suitable solvents such as, for example, methylene chloride.
Abbreviations used:
______________________________________ Fmoc 9-fluorenylmethyloxycarbonyl Ddz .alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl Bpoc 2-[4-biphenylyl]-2-propyloxycarbonyl Msc Methylsulfonylethyloxycarbonyl Peoc pyridylethyloxycarbonyl Pse phenylsulfonylethyloxycarbonyl Tse tolylsulfonylethyloxycarbonyl HONSu N-hydroxysuccinimide HOBt 1-hydroxybenzotriazole HOObt 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine THF tetrahydrofuran DMF dimethylformamide DMAP dimethylaminopyridine Acm Acetamidomethyl .epsilon.-Ahx .epsilon.-Aminohexanoyl Aoc cis, endo-2-Azabicyclo[3.3.0]octane-3-S-carbonyl Boc tert.-Butyloxycarbonyl But tert.-Butyl Bzl Benzyl Cl-z 4-Chlorobenzyloxycarbonyl Dnp 2,4-Dinitrophenyl Mac Mercaptoacetic acid Mbu Mercaptobutyric acid Me Methyl 4-Mebzl 4-Methylbenzyl Mpr Mercaptoproprionic acid Mtr 4-Methoxy-2,3,6-trimethylphenylsulfonyl Mts Mesitylene-2-sulfonyl TFA Trifluoroacetic acid Tcs 4-Methylphenylsulfonyl Trt Trityl Aad .alpha.-aminoadipic acid .gamma.Abu .gamma.-aminobutyric acid AC acetyl BHA benzhydrylamino hArg homoarginine MBHA methylbenzhydrylamino Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl Nle norleucine NMP N-methylpyrrolidone Tic 3-carboxy-1,2,3,4-tetrahydroisoquinoline ______________________________________